JPH07128317A - Data processing method for liquid chromatography - Google Patents

Abstract

PURPOSE:To correct the shift of peak time by shifting the peak time of background chromatogram containing no sample to the peak time of chromatogram existing in an arbitrarily designated time section. CONSTITUTION:A backgroud base line data is measured and stored or a data measured in the past is fetched from a memory. A predictive time band 16 for switching the butter is designated and then the analysis is started or a decision is made on whether any past specimen data is present or not. Subsequently, the analysis is started to collect specimen data which is fetched at the time of recalculation. The retention time of a peak 5 existing in a designated section 16 is then detected and compared with that of a peak 5' to be subtracted. If any difference 10 is detected, the retention time of peak 5' in the section 16 is shifted to match the peak 5. The absorbance of all background data is then subtracted from that of all chromatograms for the specimen data.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a data processing method for a liquid chromatograph.

[0002]

2. Description of the Related Art A liquid chromatograph data processor is required to accurately and beautifully output a report including a chromatogram. The subtraction function is a method to create a beautiful chromatogram. This function is used for high-sensitivity analysis, etc., when the fluctuation or increase of the baseline is large, the background baseline without sample is subtracted from the chromatogram containing the normal sample peak, and the apparently horizontal chromatogram is obtained. As a result, it is accurate and beautiful.

However, this function has the following drawbacks.
That is, when the background baseline is gentle, the subtraction is smooth and a substantially horizontal baseline can be drawn, but when there is a sudden change of different liquids, a rapid baseline change occurs. In this case, when the background baseline is subtracted, a negative peak occurs due to a slight time difference in retention time. This phenomenon not only makes the baseline correction inaccurate, but also makes the chromatogram look unattractive.

The phenomenon will be described below with reference to an actual example. FIG. 1 is an example of a chromatogram of amino acid analysis performed by stepwise switching between sodium citrate buffer solutions having different pH and Na + concentrations. An example of an amino acid analysis method is described in Japanese Patent Nos. 1517975 and 1607345.
In FIG. 1, since the data processing device electrically enlarges the scale to improve the sensitivity, the baseline shifts stepwise.

Therefore, in order to eliminate this shift increase, as shown in FIG. 2, analysis is performed without the sample under the same analysis conditions as in FIG. 1 to obtain blank data. Next, when the raw data of FIG. 2 is subtracted from the raw data of FIG. 1 by recalculating the values at each time point, a chromatogram having a flat and easy-to-see baseline is usually obtained as shown in FIG.

However, in some cases, as shown in FIG. 3, a negative peak appears between A1a and Cys, so that the baseline correction before and after that shifts to the negative side to calculate an inaccurate area. However, it often happens.

[0007]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a data processing method which can improve the essential problems of these liquid chromatographs in terms of data processing and can perform accurate subtraction. It is in.

[0008]

[Means for Solving the Problems] The cause of this problem will be described below with reference to the drawings.

FIG. 5 shows a part of a chromatogram obtained by an amino acid analyzer. The x-axis shows time and the y-axis shows absorbance. Amino acid analysis mainly separates about 20 amino acids by elution by switching a plurality of buffer solutions having different pH and Na + concentration by stepwise (gradient) or gradient method. In this case, Gly1, A1
a2 is eluted in the second buffer region 6, but Cys3, V
Al4 can be well separated by elution in the third buffer region 7. However, here, at the boundary 14 of switching, the step 8 of the baseline is generated. This step 8 is caused by the difference in the background (absorbance) of the second buffer solution 6 and the third buffer solution 7 with respect to the wavelength of 570 nm, and becomes more prominent as the sensitivity becomes higher.
The peak 5 at the time of switching is a residue or polymer compound mainly eluted from the ion-exchange resin of the column and does not come from the sample. Base line 9 indicates the electrical zero point in this case.

Next, FIG. 6 shows a chromatogram obtained by sampling pure water containing no sample, for example, pure water by the same method as these analysis methods. It is ideal that the shaded area floats just like the baseline 15 in FIG. Therefore, the floating step 8'due to the switching of the buffer solution also appears as in FIG. Then, from the chromatogram of FIG.
If you subtract the shaded area, the baseline 1
A 5'horizontal chromatogram is usually obtained. However, in reality, as shown in FIG. 7, an abnormal peak 12 and a negative peak 11 may appear at the boundary of switching the buffer solution. All of these peaks are fictitious peaks and are peaks that should never appear. The cause of the occurrence of these fictitious peaks is caused by a slight deviation 10 of the floating position (time) of the baseline as shown in FIG. These deviations 10 become more prominent when the uplift in FIG. 6 is sudden. In the stepwise switching, this may occur for several seconds due to fluctuations in the flow rate of the pump, and it is difficult to avoid it completely. If this is a gentle transition like the gradient elution method,
The baseline is as shown in FIG. 10, and when subtracted from the chromatograph, even if there is a time lag, the value is small, so it is almost horizontal and inconspicuous.

In order to achieve the above object, it is necessary to provide the data processing device with means for correcting the time shift at the time of switching.

First, as a first means, as shown in FIG. 8, the width 1 of the switching time zone of the buffer solution predicted in advance is 1
Specify 6 in advance. Then, by shifting (shifting along the time axis) the peak 5'of the peak 5'on the chromatogram (Fig. 6) to the peak 5 of the original chromatogram (see Fig. 5) to be subtracted, a shift 10 at the time of this switching is obtained. Is set to zero to match.

As a second means, as shown in FIG. 11, similarly to the first means, the width 16 'of the switching time zone is previously set.
Is specified, the rising points 17 and 17 'of the step 8 "of the baseline in this width 16' are detected, and this width 16 'is detected.
The subtracted baseline chromatogram at
Shift the upper part of the original chromatogram (see Fig. 5) to be subtracted in this width 16 ',
The shift 10 at the time of switching is set to zero.

As a third means, a width 16 "of the switching time zone is designated in advance as shown in FIG.
The baseline chromatogram (see FIG. 6), which is subtracted at the first Δt time of 6 ″, is shifted and subtracted from the original chromatogram (see FIG. 5). The value at this time is FIG. 12a).
As shown in FIG. Then Δt
By shifting and subtracting each, the minus peak 11 'becomes smaller as shown in FIG. 12b). Then, the scanning of Δt is stopped when the negative peak disappears as a result of subtraction as shown in FIG. 12d).

[0015]

The operation of each means will be described below.

The first means is effective when there are peak 5 (FIG. 5) and peak 5 '(FIG. 6) at the time of switching. That is, since the time of peak 5 at the time of switching coincides with the time of peak 5 ′, when subtracting the background baseline diagram 6 from the chromatogram diagram 5, the negative peak should be minimized as shown in FIG. You can

The second means is a method of detecting the rising point of the baseline at the time of switching as shown in FIG. 11 as shown by 17 and 17 'in FIG. This method is effective when there is no peak 5'as shown in FIG. 6 in the baseline shift at the time of switching as shown in FIG.

The third means is, as shown in FIG. 12, scanning by Δt until the negative peak does not exist in the subtracted result. In this method, scanning is performed until the number of negative peaks is minimized while searching for the presence of negative peaks, so subtraction can be performed with the least error.

The width 16 of the switching time zone of the buffer solution shown in FIG. 8, which is predicted in advance, is set so that even if the width 16 is present in the chromatogram a plurality of times, the same subtraction treatment can be performed. To do.

[0020]

EXAMPLES Examples of the present invention using the above means will be described below.

The first embodiment is described in the flow chart of FIG.

1) First, the background baseline data that is the source of the subtraction is measured and stored.
Alternatively, if necessary, similar data measured in the past is retrieved from the storage memory.

2) Designate the width 16 of the expected buffer solution switching time zone.

3) Whether to start the next analysis, or
It is determined whether or not there is sample data measured in the past.

4) Start analysis and collect sample data. Alternatively, in case of recalculation, sample data collected in the past is retrieved.

5) Next, the retention time of the peak 5 existing in the designated section 16 is detected and compared with the retention time of the peak 5'to be subtracted.

6) If there is a difference between the two retention times, the retention time of the peak 5'in the section 16 is shifted to match the retention time of the peak 5. The part where the baselines overlap due to the shift is deleted. In addition, a blank portion of the baseline due to shift is connected by a straight line.

7) Subtract calculation is performed by subtracting the absorbance height points of all background data from the absorbance height points of all chromatograms of the sample data.

8) Publish or save the results.

9) Whether all the analysis samples have been completed,
Alternatively, in the case of recalculation, it is determined whether all the recalculations have been completed.

The second embodiment is described in the flow chart of FIG. First, 1) -4) is performed as in the first embodiment.

10) A deviation 10 'between the retention times 17 and 17' at the rising point of the designated section 16 'in FIG. 11 is detected.

11) Next, the data in 16 'within the designated equivalent section of the background baseline is shifted to match the rising points 17 and 17'. Then the entire data is subtracted.

The third embodiment is described in the flow chart of FIG. First, 1) -4) is performed as in the first embodiment.

12) Subtract the background baseline data from the sample data in 16 "within the designated section in FIG.

13) The retention time of the baseline data is shifted until the negative peak falls below a certain level, the deviation is corrected, and this is repeated to detect when the negative peak falls below a certain level. Then the entire data is subtracted.

According to the above embodiment, the following effects can be obtained.

The rise of the baseline as shown in FIG. 1 is eliminated (reduced), and highly accurate data processing results can be obtained even in high sensitivity analysis.

Since the occurrence of the minus peak as shown in FIG. 3 is eliminated (decreased), the baseline shifted to the minus side is eliminated (decreased), and the correct baseline correction can be performed.

Since the baseline is horizontal, even a minute peak can be detected, and the detection sensitivity is substantially increased.

The chromatogram looks good when it is reported.

[0042]

According to the present invention, since a correct subtraction process can be carried out, a beautiful chromatogram and a correct quantitative value of a peak can be obtained.

[Brief description of drawings]

FIG. 1 is an explanatory diagram of a chromatogram for amino acid analysis showing a conventional example.

FIG. 2 is an explanatory diagram of a chromatogram of a baseline blank.

FIG. 3 is a diagram showing an example of subtraction data showing a conventional example.

FIG. 4 is a diagram showing an example of subtract data according to the present invention.

FIG. 5 is an explanatory view showing a part of a chromatogram of amino acid analysis.

FIG. 6 is an explanatory diagram of a background baseline of the above.

7 is an explanatory diagram of a chromatogram obtained by subtracting FIG. 6 from FIG.

FIG. 8 is an explanatory diagram showing a deviation of a step portion between FIGS. 1 and 2;

FIG. 9 is a view showing a case where the deviation of the step portion is corrected.
It is a chromatogram explanatory drawing at the time of subtracting.

FIG. 10 is an explanatory diagram of a background baseline in the case of gradient switching.

FIG. 11 is an explanatory diagram showing a deviation in rising of a step.

FIG. 12 is an explanatory diagram of a minus peak portion generated by subtraction of a step portion.

FIG. 13 is a flowchart showing a first embodiment.

FIG. 14 is a flowchart showing a second embodiment.

FIG. 15 is a flowchart showing a third embodiment.

[Explanation of symbols]

1 ... Gly (glycine), 2 ... Ala (alanine), 3
… Cys (cystine), 4… Val (valine), 5,
5 '... peak during switching, 6 ... second buffer solution area, 7 ... third buffer solution area, 8, 8' ... step of base line, 9 ... base line,
10 ... Shift when switching, 11 ... Minus peak, 12 ... Abnormal peak, 13 ... Background baseline, 14 ...
Boundary of switching, 15, 15 '... Baseline, 16 ... Width of switching time zone, 17 ... Rise point of step.

Claims (3)

[Claims] 1. A measurement value of a background chromatogram containing no sample under the same measurement conditions as the chromatogram, which is stored in a data processing apparatus for liquid chromatograph, from a measurement value of a chromatogram containing a sample to be measured. In a data processing device for liquid chromatograph with a subtraction function, a peak of a background chromatogram that does not include the above sample is present at the peak apex time of the chromatogram that includes the above sample that exists within the arbitrarily designated time interval. A data processing method for a liquid chromatograph, characterized in that the peak time is shifted to correct the time shift of the peak. 2. The baseline rising start-up time of a background chromatogram not containing the sample at the start-up time of the baseline rising start of the chromatogram containing the sample, which is present within an arbitrarily designated time period. A data processing method for a liquid chromatograph, characterized in that the start time is shifted to correct the deviation of the start time. 3. The baseline rise start time of the background chromatogram not including the sample, from the baseline rise start time of the chromatogram including the sample, within the arbitrarily designated time period. , So that the result of subtracting each measurement point does not become negative, or becomes the minimum negative,
At the point where the baseline rise rise start time of the background chromatogram not including the above sample is sequentially shifted afterward, and the subtraction result of each measurement point is the closest to zero,
A method for processing data for a liquid chromatograph, characterized in that the shift is stopped and each measurement point at this point is subtracted.